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Abstract

Background

Because of the lack of reproducible brainstem ischemia models in rodents, the temporal
profile of ischemic lesions in the brainstem after transient brainstem ischemia has
not been evaluated intensively. Previously, we produced a reproducible brainstem ischemia
model of Mongolian gerbils. Here, we showed the temporal profile of ischemic lesions
after transient brainstem ischemia.

Results

Brainstem ischemia was produced by occlusion of the bilateral vertebral arteries just
before their entry into the transverse foramina of the cervical vertebrae of Mongolian
gerbils. Animals were subjected to brainstem ischemia for 15 min, and then reperfused
for 0 d (just after ischemia), 1 d, 3 d and 7 d (n = 4 in each group). Sham-operated
animals (n = 4) were used as control. After deep anesthesia, the gerbils were perfused
with fixative for immunohistochemical investigation. Ischemic lesions were detected
by immunostaining for microtubule-associated protein 2 (MAP2). Just after 15-min brainstem
ischemia, ischemic lesions were detected in the lateral vestibular nucleus and the
ventral part of the spinal trigeminal nucleus, and these ischemic lesions disappeared
one day after reperfusion in all animals examined. However, 3 days and 7 days after
reperfusion, ischemic lesions appeared again and clusters of ionized calcium-binding
adapter molecule-1(IBA-1)-positive cells were detected in the same areas in all animals.

Conclusion

These results suggest that delayed neuronal cell death took place in the brainstem
after transient brainstem ischemia in gerbils.

Background

In the central nervous system, certain areas are selectively damaged even after a
brief ischemic insult, and this topographical heterogeneity is known as "selective
vulnerability of the brain". Hippocampal CA1 and neocortical III, V, and VI are extremely
vulnerable to ischemia and hypoxia [1]. The mechanism responsible for this vulnerability is of particular importance to
establish therapeutic procedures, because elucidation of the mechanism may lead to
the development of novel therapy to ameliorate ischemic damage.

Pathologic aspects and the topographic distribution of ischemic lesions after transient
ischemia have been extensively studied in the rodent forebrain [2,3]. However, little is known about the distribution of ischemic lesions after transient
brainstem ischemia because of the lack of reproducible brainstem ischemia models in
rodents. Previously, we established a brainstem ischemia model in gerbils by occlusion
of the bilateral vertebral arteries, and demonstrated selective vulnerability after
permanent brainstem ischemia [4]. This gerbil model has the following advantages: (1) it produces brainstem ischemia
without intracranial injury, (2) it produces severe, reproducible brainstem ischemia,
and (3) it allows reperfusion.

In the present study, using this animal model, we investigated the temporal profile
of ischemic lesions in the brainstem after transient brainstem ischemia in gerbils.
We demonstrated ischemic lesions by immunostaining for microtubule-associated protein
2 (MAP2) in the lateral vestibular nucleus and the ventral part of the spinal trigeminal
nucleus three days after transient brainstem ischemia, while these ischemic lesions
were not found one day after ischemia. This delayed neuronal damage in the brainstem
is reminiscent of the delayed neuronal cell death in the hippocampus after transient
forebrain ischemia [5].

Methods

Animals and surgical procedure

Adult 12-16 week-old male Mongolian gerbils, weighing 60-80 g, were used in this study.
All experiments were approved by the Ethics Committee of Ehime University Graduate
School of Medicine and were conducted according to the Guidelines for Animal Experimentation
at Ehime University Graduate School of Medicine. The gerbils were housed in an animal
room with a temperature of 21 to 23°C and a 12-hour light/dark cycle (light on: 7
a.m. to 7 p.m.). The animals were allowed free access to food and water until the
end of the experiment.

The gerbils were randomly divided into four groups, which were subjected to brainstem
ischemia for 15 min and reperfused for 0 d (just after ischemia), 1 d, 3 d and 7 d
(n = 4 in each group). Sham-operated animals (n = 4) were used as control. Animals
were anesthetized with 1% halothane in 70% N2O and 30% O2. Anesthetized animals were orotracheally intubated with a ventilation tube. To facilitate
access to the vertebral arteries, animals were placed in the supine position on a
table tilted at approximately 30° to the horizontal. An anterior midline cervical
incision was made, and the musculi longus colli were dissected to expose the vertebral
arteries just before their entry into the transverse foramina of the cervical vertebrae.
Both vertebral arteries were looped with 4-0 silk sutures. Then, the suture around
each vertebral artery was pulled by a 5-g weight to occlude the circulation for 15
min. Consequently, apnea was observed within 1 min after occlusion, and subsequent
convulsions were observed in all four limbs for about 1 min. After convulsions had
ceased, all animals became unresponsive and lost their corneal reflex. Mechanical
ventilation was initiated immediately after apnea was elicited during ischemia. The
tidal volume was set to 1 ml and the rate was set to 70 breaths per minute. After
15 min of ischemia, the sutures were cut and removed to allow recirculation, which
was confirmed by visual observation through an operating microscope. Within 10 min
after reperfusion, spontaneous breathing reappeared and mechanical ventilation was
ceased within 15 min after reperfusion.

Rectal temperature was maintained between 36.5 and 37.0°C by a heating lamp and a
heating pad connected to a thermistor (ATB-1100, Nihon Koden, Tokyo, Japan) during
surgery and until 1 h after reperfusion. After resuscitation, the animals were maintained
in an air-conditioned room at about 22°C.

Histological procedures

After deep anesthesia with a lethal dose of sodium pentobarbital (0.1 g/kg), the gerbils
were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and the
brain was dissected out. After fixation with the same fixative for overnight the brain
was dehydrated and embedded in paraffin. To investigate the temporal profile of ischemic
lesions in the brainstem, we performed immunostaining for MAP2, IBA-1 and GFAP at
the level of the lateral vestibular nucleus in the brainstem (5.5 mm caudal to the
bregma) since this area has been reported to be most vulnerable to ischemia [4]. Coronal 5-μm-thick sections were examined by immunostaining for microtubule-associated
protein 2 (MAP2), IBA-1 and glial fibrillary acidic protein (GFAP). Sections were
immunostained using a Vectastain ABC Elite Kit (Vector Laboratories; Burlingame, Calif)
with polyclonal anti-MAP2 (donated by Dr. Niinobe, Osaka University), polyclonal anti-IBA-1
(019-19741, Wako, Osaka, Japan) or monoclonal anti-GFAP (G9369, Sigma, St. Louis,
USA) antibodies. Endogenous peroxidase in deparaffinized tissue sections was blocked
for 10 minutes with 3% H2O2 in deionized water, followed by blocking with 10% goat serum diluted in 0.2% Tween-20
in phosphate buffered saline at room temperature for 1 hour. The tissues were then
incubated with primary antibody (anti-MAP2, 1:1000; anti-IBA-1, 1:500; anti-GFAP,
1:500) at 4°C overnight. Tissue sections were washed and incubated with secondary
antibody (1:1000) for 1 hour at room temperature. After washing, sections were incubated
with ABC complex for 30 minutes at room temperature, and then stained with the chromogenic
substrate 3, 3-diaminobenzidine tetrahydrochloride (DAB) and H2O2, until optimal staining was obtained.

Measurement of ischemic lesions

MAP2-stained sections were analyzed and images were viewed using a microscope (ECLIPSE
E800, Nikon, Tokyo, Japan). The ischemic lesions detected by the loss of immunoreaction
for MAP2 were measured and quantification was performed on images using ImageJ software
(National Institutes of Health, Bethesda, MD).

Statistics

All values are given as mean ± SD. Statistical analysis was performed with the Statistical
Package for the Social Sciences, release 15 (SPSS ver. 15). Differences were analyzed
using one-way ANOVA followed by Bonferroni's multiple comparison test. A p value of
less than 0.05 was considered to indicate statistical significance.

Results

Immunohistochemical investigation

Four gerbils each were used for the reperfusion periods of 0, 1 d, and 3 d. As for
the reperfusion period of 7 d, we evaluated three animals because one animal died
of respiratory failure 5 days after ischemia. Sham-operated animals (n = 4) were used
as control. Loss of immunoreaction for MAP2 in neuropils, nerve cell bodies, and dendrites
was used as the criterion for the presence of ischemic lesions. The findings were
compared with those in sham-operated controls. Each brain section was examined by
two investigators; and whenever there was any uncertainty, a third investigator examined
the specimen without any prior information.

Just after brainstem ischemia

Ischemic lesions detected by immunostaining for MAP2 were found in the lateral vestibular
nucleus (LVe; blue arrows in Figure 1B) and the ventral part of the spinal trigeminal nucleus (Sp5; red arrows in Figure
1B) in all 4 animals (100%). Higher magnification photomicrographs of ischemic lesions
showed loss of immunoreaction for MAP2 in neuropils and nerve cell bodies in LVe (blue
arrows in Figure 2B) and the ventral part of Sp5 (red arrows in Figure 3B). Compared with sham-operated controls, there was no change in IBA-1 (a marker of
microglia and monocytic lineage) and GFAP (a marker of astrocytes) expression (Figure
1G and 1L).

Figure 1.Representative photomicrographs of immunostaining after transient brainstem ischemia
for 15 min (pons). Left column shows immunoreactivity for MAP2 (A-E); middle column shows immunoreactivity
for IBA-1 (F-J); right column shows immunoreactivity for GFAP (K-O). Ischemic lesions
with loss of MAP2 staining were detected in the lateral vestibular nucleus (LVe: B;
blue arrows) and the ventral part of the spinal trigeminal nucleus (Sp5: B; red arrows)
after bilateral vertebral artery occlusion (BVO) for 15 min. These ischemic lesions
had disappeared at 1 day after reperfusion (C). At 3 days and 7 days after reperfusion,
ischemic lesions appeared again and expanded further (D and E; blue and red arrows).
New ischemic lesions were detected in the dorsal part of Sp5 (D and E; blue arrowheads)
and ventral cochlear nucleus (VC) (D and E; red arrowheads). At the same time, clusters
of amoeboid microglia/macrophages were detected in the same areas (I and J; blue and
red arrows/arrowheads). At 3 days and 7 days after reperfusion, immunoreactivity for
GFAP was lost in the ischemic lesions, and increased immunoreactivity for GFAP was
detected around ischemic lesions (N and O; blue and red arrows/arrowheads). Scale
bar = 1 mm.

Figure 2.Representative photomicrographs of immunostaining after transient brainstem ischemia
for 15 min (vestibular nucleus). Left column shows immunoreactivity for MAP2 (A-E); middle column shows immunoreactivity
for IBA-1 (F-J); right column shows immunoreactivity for GFAP (K-O). Ischemic lesions
with loss of immunoreactivity for MAP2 were seen in the lateral vestibular nucleus
after bilateral vertebral artery occlusion (BVO) for 15 min (B; blue arrows), and
these lesions had disappeared at 1 day after reperfusion (C). However, ischemic lesions
had reappeared and expanded further at 3 days and 7 days after reperfusion (D and
E; blue arrows). Clusters of IBA-1-positive amoeboid microglia/macrophages (I and
J; blue arrows) and loss of expression of GFAP (N and O; blue arrows) were detected
in the same areas where MAP2 expression was markedly lost at 3 days and 7 days after
reperfusion. Increased immunoreactivity for GFAP (N and O; blue arrows) was also detected
around ischemic lesions at 3 days and 7 days after reperfusion. Scale bars = 0.5 mm.

Figure 3.Representative photomicrographs of immunostaining after transient brainstem ischemia
for 15 min (spinal trigeminal nucleus). Left column shows immunoreactivity for MAP2 (A-E); middle column shows immunoreactivity
for IBA-1 (F-J); right column shows immunoreactivity for GFAP (K-O). Ischemic lesions
with loss of immunoreactivity for MAP2 were seen in the ventral part of Sp5 after
bilateral vertebral artery occlusion (BVO) for 15 min (B; red arrows), and these lesions
had disappeared at 1 day after reperfusion (C). However, ischemic lesions had reappeared
and expanded further (D and E; red arrows) and new ischemic lesions were detected
in the dorsal part of Sp5 (D and E; blue arrowheads) at 3 days and 7 days after reperfusion.
Clusters of IBA-1-positive amoeboid microglia/macrophages (I and J; red arrows and
blue arrowheads) and loss of GFAP expression (N and O; red arrows and blue arrowheads)
were detected in the same areas where MAP2 expression was lost at 3 days and 7 days
after reperfusion. Increased immunoreactivity for GFAP (N and O; red arrows and blue
arrowheads) was also detected around ischemic lesions at 3 days and 7 days after reperfusion.
Scale bars = 0.5 mm.

One day after brainstem ischemia

No ischemic lesion was detected by MAP2 staining (Figure 1C). Furthermore, there was no change in IBA-1 and GFAP expression, compared with that
in sham-operated controls (Figure 1H and 1M).

Three days after brainstem ischemia

Ischemic lesions in LVe (blue arrows in Figures 1D and 2D) and the ventral part of Sp5 (red arrows in Figures 1D and 3D) appeared again in all 4 animals (100%). Compared with the ischemic lesions just
after brainstem ischemia, ischemic lesions in LV expanded ventrally to include the
spinal vestibular nucleus (SpVe) in 2 out of 4 animals (50%). De novo ischemic lesions
were detected in the dorsal part of Sp5 (blue arrowheads in Figures 1D and 3D) and ventral cochlear nucleus (VC) (red arrowheads in Figures1D and 4D) in 2 out of 4 animals (50%).

Figure 4.Representative photomicrographs of immunostaining after transient brainstem ischemia
for 15 min (cochlear nucleus). Left column shows immunoreactivity for MAP2 (A-E); middle column shows immunoreactivity
for IBA-1 (F-J); right column shows immunoreactivity for GFAP (K-O). Ischemic lesions
with loss of immunoreactivity for MAP2 were seen in the ventral cochlear nucleus (VC)
at 3 days and 7 days after reperfusion (D and E; red arrowheads). Clusters of IBA-1-positive
amoeboid microglia/macrophages (I and J; red arrowheads) and loss of GFAP expression
(N and O; red arrowheads) were detected in the same areas where MAP2 expression was
lost at 3 days and 7 days after reperfusion. Increased immunoreactivity for GFAP (N
and O; red arrowheads) was also detected around ischemic lesions at 3 days and 7 days
after reperfusion. Scale bars = 0.5 mm.

In addition, IBA-1 immunoreactivity was markedly up-regulated in the central part
of the ischemic lesions where MAP2 immunostaining was lost. Up-regulation of IBA-1
immunoreactivity was detected in LVe (blue arrows in Figures 1I and 2I) and the ventral part of Sp5 (red arrows in Figures 1I and 3I) in 3 out of 4 animals (75%). Up-regulation of IBA-1 immunoreactivity was also detected
in the dorsal part of Sp5 (blue arrowheads in Figures 1I and 3I) and ventral cochlear nucleus (VC) (red arrowheads in Figures 1I and 4I) in 2 out of 4 animals (50%). Higher magnification photomicrographs demonstrated
strongly IBA-1-positive cells in these areas. These IBA-1-positive cells displayed
an amoeboid shape including only small perisomal lamellopodial expansions or a few
unbranched processes. They were morphologically easily distinguishable from ramified
microglial cells, which were recognized by their thick processes and large cell bodies.

Furthermore, immunoreactivity for GFAP disappeared in ischemic lesions where immunostaining
for MAP2 was lost, whereas immunoreactivity for GFAP increased in the neighboring
areas around ischemic lesions. A reduction of GFAP staining was detected in LVe (blue
arrows in Figures 1N and 2N) and the ventral part of Sp5 (red arrows in Figures 1N and 3N) in 3 out of 4 animals (75%). A reduction of GFAP staining was also detected in the
dorsal part of Sp5 (blue arrowheads in Figures 1N and 3N) and the ventral cochlear nucleus (VC) (red arrowheads in Figures 1N and 4N) in 2 out of 4 animals (50%). Higher magnification photomicrographs showed that GFAP-positive
astrocytes were not observed in ischemic lesions where immunostaining for MAP2 was
lost. Reactive astrocytes with thick, long GFAP-positive processes were distributed
around ischemic lesions.

Seven days after brainstem ischemia

Ischemic lesions detected by immunostaining for MAP2 expanded further (Figure 5A-D). Ischemic lesions in LVe (blue arrows in Figures 1E and 2E) and the ventral part of Sp5 (red arrows in Figures 1E and 3E) appeared in all 3 animals (100%). Ischemic lesions were also detected in the dorsal
part of Sp5 (blue arrowheads in Figures 1E and 3E) and the ventral cochlear nucleus (VC) (red arrowheads in Figures 1E and 4E) in 1 out of 3 animals (33%).

Figure 5.Incidence maps of immunoreactivity for MAP2 (decrease) and IBA-1(increase) in coronal
gerbil brain sections at various times after transient brainstem ischemia for 15 min
(at level of pons [5.5 mm caudal to bregma]). Areas of altered immunoreactivity were outlined in all animals examined and superimposed
to represent the incidence map (%). For details, see text.

IBA-1 immunoreactivity was markedly up-regulated in ischemic lesions where MAP2 immunostaining
was lost. Compared with the profile of IBA-1 staining three days after brainstem ischemia,
strongly IBA-1-positive cells with an amoeboid shape were distributed more peripherally
in ischemic lesions as well as in the center of ischemic lesions. Up-regulation of
IBA-1 immunoreactivity was detected in LVe (blue arrows in Figures 1J and 2J) and the ventral part of Sp5 (red arrows in Figures 1J and 3J) in all three animals (100%). Up-regulation of IBA-1 immunoreactivity was also detected
in the dorsal part of Sp5 (blue arrowheads in Figures 1J and 3J) and ventral cochlear nucleus (VC) (red arrowheads in Figures 1J and 4J) in one out of three animals (33%).

GFAP immunoreactivity disappeared in the central part of ischemic lesions where MAP2
immunostaining was lost. Immunoreactivity for GFAP increased in the periphery of ischemic
lesions as well as the neighboring areas around ischemic lesions. These results suggested
that reactive astrocytes proliferated in the neighboring areas around ischemic lesions
and migrated into the ischemic lesions. A reduction of GFAP staining was detected
in LVe (blue arrows in Figures 1O and 2O) and the ventral part of Sp5 (red arrows in Figures 1O and 3O) in all 3 animals (100%). A reduction of GFAP staining was also detected in the dorsal
part of Sp5 (blue arrowheads in Figures 1O and 3O) and the ventral cochlear nucleus (VC) (red arrowheads in Figures 1O and 4O) in 1 out of 3 animals (33%).

Temporal profile of ischemic lesions

The total area of ischemic lesions detected by MAP2 staining in each animal was calculated
and summarized in Figure 6. Just after brainstem ischemia for 15 min, the total area of ischemic lesions was
0.33 ± 0.041 [Mean ± SD] (mm2). Although ischemic lesions disappeared one day after brainstem ischemia, evolution
of ischemic lesions was detected 3 and 7 days after transient brainstem ischemia (0.42
± 0.034 and 0.76 ± 0.064, respectively).

Figure 6.Temporal profile of ischemic lesions detected by MAP2 staining after transient brainstem
ischemia for 15 min. The area of ischemic lesions detected by MAP2 staining in each animal was calculated.
For details, see text. C: control, 0 d: just after brainstem ischemia for 15 min,
1 d, 3 d and 7 d: 1, 3 and 7 days after transient brainstem ischemia for 15 min, respectively.
All values are given as mean ± SD. Differences were analyzed using one-way ANOVA followed
by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered
to indicate statistical significance. ** and $$ indicate significant (p < 0.01) difference
vs. C and 0 d, respectively. $ indicates significant (p < 0.05) difference vs. 0 d.

Discussion

Detection of morphological damage in early cerebral ischemia is difficult with conventional
histological procedures including triphenyltetrazolium chloride and hematoxylin-eosin
staining. With these conventional methods, morphological evidence of neuronal death
does not become apparent until 1 to 2 hours after the onset of cerebral ischemia.
However, early ischemic lesions can now be detected by applying immunohistochemical
methods, and a reduction in microtubule-associated protein 2 (MAP2) immunoreactivity
has been found to be an early, sensitive marker of ischemic neuronal damage [6]. In our study, by using this method, we showed that the lateral vestibular nucleus
(LVe) and the ventral part of the spinal trigeminal nucleus (vSp5) were particularly
vulnerable to ischemia.

In the LVe, multipolar giant neurons (Deiter's neurons) were most vulnerable to ischemia.
Vestibular neurons receive excitatory glutaminergic input from the vestibular nerve
[7] and commissural excitatory afferents [8]. Immunohistochemical and in situ hybridization histochemical studies revealed the
highest glutamate receptor 2 (GluR2) expression in giant Deiter's neurons of the lateral
vestibular nucleus and the lowest expression in small neurons throughout the vestibular
nuclei [9]. These reports suggest that Deiter's neurons receive excitatory input and have selective
sensitivity to excitotoxicity. As an analogy to hippocampal neurons [10], we speculate that an ischemia-induced alteration of GluR2 expression in Deiter's
neurons induced cell death. Although further investigations are required to clarify
the mechanisms underlying this selective vulnerability and delayed neuronal damage,
they may also be related to several other factors such as the degree of cerebral hypoperfusion
after reperfusion [11], inhibition of protein synthesis [12], neutrophil infiltration following reperfusion [13], free radical production [14], dysfunction of the mitochondrial shuttle system [15] or apoptosis [16].

Furthermore, we showed that the ischemic lesions in LVe and vSp5 had disappeared one
day after reperfusion, but appeared again three days after reperfusion and thereafter.
The observed loss of immunoreaction for MAP2 may reflect cytoskeletal breakdown, because
MAP2 is involved in maintaining the structural integrity of the neuronal cytoskeleton
[17]. The ischemia-induced rapid elevation of intracellular Ca2+ concentration and subsequent activation of Ca2+-dependent phosphatases (e.g., calcineurin) and proteases (e.g., calpains) can lead
to dephosphorylation and proteolytic degradation of MAP2 [18,19]. Therefore, ischemia-induced loss of immunoreaction for MAP2 is considered to be
a reliable marker of neurons that are already undergoing irreversible processes in
cell death [20]. However, Kitagawa et al. showed that loss of MAP2 immunostaining preceded the development
of overt neuronal loss in a gerbil model of transient forebrain ischemia [6]. Our results are also consistent with this notion that MAP2 immunostaining can be
used as an indicator of still viable neurons that will undergo irreversible injury
only at a later time point.

We also demonstrated clusters of IBA-1-expressing cells in the ischemic lesions where
MAP2 staining was lost three days after ischemia and thereafter. The rat Iba1 gene has been identified as a microglia-specific transcript [21]. The isolated Iba1clone was 0.8 kb, a rather small cDNA encoding a 17-kDa protein consisting of 147
amino acids. IBA-1 is an interferon-γ (IFN-γ)-inducible Ca2+-binding EF-hand protein that is encoded within the HLA class III genomic region.
Expression of IBA-1 is mostly limited to the monocyte/macrophage lineage, and is augmented
by cytokines such as IFN-γ. It was assumed that IBA-1 is a novel molecule involved
in inflammatory responses and allograft rejection, as well as activation of macrophages
[22]. In the normal brain, IBA-1 is highly expressed in resident microglial cells, but
is never expressed in neurons and astrocytes [22]. After ischemia, IBA-1 is also expressed in activated resident microglial cells and
infiltrating hematogenous macrophages [23,24].

Resident microglial cells rapidly became activated after ischemia. They developed
amoeboid or rounded cell bodies and migrated rapidly into the ischemic lesion. For
example, IBA-1 expression was rapidly up-regulated in the gerbil hippocampal CA1 region
at 30 min after transient forebrain ischemia for 5 min [25]. However, microglial cells did not proliferate rapidly. Denes et al. showed that
resident microglial cells exhibited intense proliferation at 48 and 72 h after transient
occlusion of the middle cerebral artery (MCA) in the mouse. Average microglial cell
number in the ischemic lesion did not increase significantly up to 48 h after transient
ischemia [26]. We also demonstrated that a significant increase in Iba-1-positive cells was not
detected in the ischemic cortex of the rat until one day after permanent MCA occlusion
(MCAO), while a significant decrease in Iba-1-positive cells was detected even 2 h
after permanent MCAO[27]. Furthermore, infiltrating hematogenous macrophages do not appear in the brain within
one day after ischemia [24]. In this study, we did not detect clusters of IBA-1-expressing cells (i.e. topical
proliferation of microglial cells) within one day after ischemia. Clusters of IBA-1-expressing
cells initially appeared in the core of the ischemic lesions three days after ischemia,
and these IBA-1-positive cells exhibited round cell bodies and possessed pseudopodia
and thin filopodia-like processes, indicating a motile phagocytic phenotype. At seven
days after ischemia, IBA-1-expressing cells with an amoeboid shape were distributed
more peripherally in the ischemic lesions as well as in the core of the ischemic lesions.
Based on their morphological features and the temporal profile of the distribution
of IBA-1-positive cells in ischemic lesions, we speculated that these IBA-1-expressing
cells were of hematopoietic origin, although we could not exclude the possibility
that they were of resident microglial origin.

In addition, we showed that delayed progression of ischemic neural death took place
in the brainstem. Although other morphological and biochemical investigations including
electron microscopic study are required for further analysis, this delayed neuronal
damage in the brainstem is reminiscent of the delayed neuronal death in the hippocampus
after transient forebrain ischemia [5]. There is increasing evidence that microglial cells contribute to delayed neuronal
death. Recruitment and activation of microglial cells gradually increase within the
hippocampal CA1 area over 24 h after transient forebrain ischemia, before the degeneration
of neurons [28]. Endangered neurons can release proinflammatory chemokines such as monocyte chemoattractant
protein-1 (MCP-1/CCL2) and secondary lymphoid-tissue chemokine (SLC/CCL21). Expression
of MCP-1 and SLC is increased in neurons after ischemia [29,30]. Subsequently, recruited and activated microglial cells produce inflammatory mediators,
including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide
(NO), which contribute to delayed neuronal death [31]. Moreover, immunosuppressants, such as FK506, prevent microglial activation and neuronal
damage after ischemia [32]. Consistent with these findings, our results also suggest that activated microglia/macrophages
play a crucial role in this delayed neuronal cell death in the brainstem.

Conclusions

In conclusion, we evaluated the evolution of ischemic lesions in the brainstem after
transient brainstem ischemia in gerbils. Using immunostaining for MAP2, ischemic lesions
were detected in LVe and vSp5 in all four animals. These ischemic lesions disappeared
one day after reperfusion, but appeared again three days after reperfusion and thereafter
in all animals examined. In addition, clusters of activated microglia/macrophages
were detected in these ischemic lesions three days after ischemia and thereafter.
These results suggest that delayed neuronal cell death took place in the brainstem
after transient brainstem ischemia in gerbils.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

The original concept was by RH, MS and KG. Animal experiments were performed by ST
and TY. Immunostaining was performed by FC and PZ. Evaluation of immunostaining was
performed by FC, RH and NH. The manuscript was written and edited by FC and RH. All
authors read and approved the final manuscript.

Acknowledgements

This project was supported, in part, by grants from the Ministry of Education, Science,
Sports and Culture of Japan. We are grateful for the secretarial assistance of Ms.
K. Hiraoka.